Hi Xianchi, How did you treat the control flow cell? And what is the composition of the control buffer that you use to disrupt binding? Is it denaturing or a mild condition?
If the Rmax=150 and Koff=1e-6, then at 7200 s (2 hr), the signal will only drop 1RU from 150RU (assuming you can reach the Rmax), is that what you see on the dissociation curve? In reality, when loading analyte at 100nM, assuming you have Kon=1e4, Koff=1e-6, binding for 30min would only let you reach ~125 RU, and at the lower concentrations will be much worse. Consequently the total dissociation after 7200s would be less than 1 RU on most curves - I am not sure if the Biacore 3000 will have a stable enough baseline for you to confidently measure that. If you try to fit the dissociation phase alone to get the Koff, the reported Koff won't be too accurate due to the low S/N ratio. On the other hand the 5-100nM spanning would generate significant changes on the binding phase, thus the global fitting should be able to get a relatively accurate KD. 0.2nM from SPR and 1nM from in-solution experiment also sounds reasonable. Zhijie From: xianchi dong Sent: Wednesday, February 20, 2013 5:45 PM To: [email protected] Subject: Re: [ccp4bb] off topic question BIAcore problem Thanks a lot for the kind reply. I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I have different truncations of my protein which behaved similarly. I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used was from 100 nM to 5 nM. I used a control buffer which can disrupt the binding. In that buffer, no binding was observed. I also used fluorescence anisotropy to measure the Ki of the interaction which showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong <[email protected]> wrote: Dear all, Recently I have measure a set of kinetic data of a receptor ligand interaction using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) .I repeat several time which give very similar results. So I am wondering if the BIAcore can measure such a low off-rate kinetic. What is the limitation of BIAcore? Any review about that? Thanks in advance. Xianchi Dong Research Fellow Children's Hospital Boston Harvard Medical School
